Albumin elisa kit

Urine creatinine was measured using a Creatinine Assay Kit (R&D Systems, Minneapolis, MN), following the manufacturer’s instructions. Total protein in urine samples was measured, as previously described (PMID: 26213685). Briefly, proteins were precipitated from urine samples by adding 40 μL of water and 200 μL of prechilled acetone to 10 μL of urine. Samples were then incubated at −20°C for 30 min and then centrifuged at 14,000 g, 4°C, for 15 min. Pellets were resuspended in 40 μL of water, and protein concentration was measured using the Pierce BCA Assay (Thermo Scientific, Rockford, IL). Albumin from urine samples was measured using the Albumin ELISA Kit (Bethyl Laboratories, Montgomery, TX), following the manufacturer’s instructions.
Acute kidney injury (AKI) biomarker levels were analyzed by multiplex assays, using the Luminex technology. The multiplex kidney injury panels (MKI1MAG-94K, MKI2MAG-94K; Merck Millipore) were used, according to the manufacturer’s instructions, to measure levels of β-2-microglobulin (B2M), KIM-1, vascular endothelial growth factor (VEGF), CysC, epidermal growth factor (EGF), lipocalin-2-NGAL, clusterin, and osteopontin (OPN). The results were read using a Bio-Plex MAGPIX Multiplex Reader and analyzed with Bio-Plex Manager 6.1 software (Bio-Rad, France)

ZDF rat kidney and urine samples were provided by Epigen Inc. to verify that kidney spatial adenine correlated with the targeted urine adenine assay. C57BL/6J, db/m, and db/db mice were obtained from The Jackson Laboratory. C57BL/6J mice were administered adenine for 4 weeks in drinking water before sacrifice, and tissues and blood samples were harvested at University of Texas Health Science Center at San Antonio. db/m and db/db mice were administered vehicle or MTDIA MTAP inhibitor for a period of 8 weeks from week 10 to week 18. An Albumin ELISA kit (catalog E101 and E90-134, Bethyl Laboratories Inc.) and creatinine colorimetric kit (catalog ADI-907-030A, Enzo Life Sciences Inc.) were used for the urinary ACR. Serum cystatin C was measured by the Quantikine ELISA kit (catalog MSCTC0, R&D Systems). Plasma creatinine and metabolites in kidney tissue were measured by Zip-Chip-mass spectrometry as previously described (12 (link)). Urine and kidney KIM-1 was measured by ELISA (catalog DY1817, R&D System).

Morning urine spot was collected bi-weekly. The urine albumin content was measured by ELISA and urinary creatinine was measured by an assay based on the Jaffe method, using albumin ELISA kit from Bethyl Lab. (#E90-134; TX, USA) and creatinine kit from Stanbio (#0420-500; TX, USA). Values are expressed as microgram albumin per milligram creatinine.

Animals were anaesthetized by intraperitoneal administration of 80 mg/kg bodyweight ketamine (Anesketin, Dechra Veterinary Products, Aulendorf, Germany), 5 mg/kg bodyweight xylazine (Rompun, Leverkusen, Germany) and 2 mg/kg bodyweight midazolam. After cessation of pain reflexes, the abdomen was opened and the abdominal aorta incised. Afterwards, a tracheotomy was carried out and the chest opened. For harvest of broncho-alveolar lavage (BAL) fluid three aliquots of 1 mL 0.9% sodium chloride solution were successively instilled and suctioned out of the lung. The range of recovery per lung was 2–2.5 mL. In order to separate cellular components and debris from fluid, a centrifugation with 1000g was carried out for 10 min. The supernatant was removed and snap frozen in liquid nitrogen and stored at –80 °C till further assessments of protein and albumin concentration as described previously [72 (link)]. In brief, albumin concentrations in BAL fluid were assessed with the albumin ELISA kit (Bethyl Laboratories, Montgomery, AL) according to the manufacturer’s instructions. Protein and albumin concentrations in BAL fluid served as indicators of vascular leak.

For glycogen storage analysis, Periodic acid–Schiff (Muto Pure Chemical, Tokyo, Japan) staining was performed on paraffin slides of iHLOs or iHEAs following the manufacturer’s instructions. For low-density lipoprotein (LDL) uptake assays, DiI-ac-LDL (Biomedical Technologies, Massachusetts, USA) was added the culture medium (200 ng/mL) and incubated for 4 h at 37 °C and 5% CO₂. After washing with PBS three times, cells were imaged by fluorescent microscopy (Leica, Wetzlar, Hesse, Germany) for LDL uptake analysis. For CYP P450 activity assays, MEFs, 2D-iHs, 2D-iHEs, iHLOs, and iHEAs were placed in an iHEA medium without dexamethasone supplement. We treated 2 μM of 3-methylcholanthrene (3-MC) CYP inducers for 72 h. The fresh medium containing 3-MC was replaced every 24 h. Dimethyl sulfoxide (DMSO) was used as a control. The P450-Glo CYP1A2 assay system (Promega, Madison, WI, USA) was used according to a modified manufacturer’s protocol. The luminescence was measured with a GLOMAX 96 micrometer luminometer (Promega, Madison, WI, USA). For the quantification of albumin secretion and urea production, the amounts of albumin and urea in the culture media were analyzed using the albumin ELISA Kit (Bethyl Laboratories, Waltham, MA, USA) and urea kit (BioAssay systems, Hayward, CA, USA), respectively, according to the manufacturer’s instructions.